Heather West Greenlee M, Uemura Etsuro, Carpenter Susan L, Doyle Robert T, Buss Janice E
Department of Biomedical Sciences, Iowa State University, Ames, Iowa 50011-1250, USA.
J Neurosci Res. 2003 Aug 15;73(4):518-25. doi: 10.1002/jnr.10684.
The distribution of glucose transporters at the cell surface has a major impact on cellular glucose uptake. In muscle cells and adipocytes, this distribution is under the control of insulin; however, neuronal glucose uptake is not acutely regulated by insulin. Factors that affect the translocation of the neuronal glucose transporter isoform GLUT3 vesicles to and their fusion with the plasma membrane are not well understood. We report that GLUT3 in PC12 cells colocalizes with SNARE complex proteins SNAP-25 and syntaxin 1, suggesting that fusion of GLUT3-containing vesicles with the plasma membrane is mediated by these proteins. In addition, it seems that GLUT3 vesicle fusion is regulated, as depolarization increases GLUT3 insertion into the plasma membrane. To study the dynamics of GLUT3 vesicle trafficking, we have created a GLUT3-GFP fusion protein that is easily expressed in PC12 cells. Trafficking of GLUT3-GFP seems normal, as 1). its distribution is similar to endogenous GLUT3, 2). GLUT3-GFP containing vesicles fuse with the plasma membrane evidenced by labeling of the fusion protein with an antibody directed against the exofacial epitope of GLUT3, and 3). glucose uptake is similar to PC12 cells not transfected with GLUT3 fusion protein. These studies are the first to examine GLUT3 trafficking and fusion in PC12 cells, and establish a model system to study regulation of the neuronal glucose transporter.
细胞表面葡萄糖转运蛋白的分布对细胞葡萄糖摄取有重大影响。在肌肉细胞和脂肪细胞中，这种分布受胰岛素控制；然而，神经元葡萄糖摄取不受胰岛素的急性调节。影响神经元葡萄糖转运蛋白异构体GLUT3囊泡向质膜转运及其与质膜融合的因素尚不清楚。我们报告，PC12细胞中的GLUT3与SNARE复合体蛋白SNAP-25和 syntaxin 1共定位，这表明含GLUT3的囊泡与质膜的融合是由这些蛋白介导的。此外，GLUT3囊泡融合似乎受到调节，因为去极化会增加GLUT3插入质膜。为了研究GLUT3囊泡运输的动力学，我们构建了一种易于在PC12细胞中表达的GLUT3-GFP融合蛋白。GLUT3-GFP的运输似乎正常，因为：1）其分布与内源性GLUT3相似；2）含有GLUT3-GFP的囊泡与质膜融合，这可通过用针对GLUT3外表面表位的抗体标记融合蛋白来证明；3）葡萄糖摄取与未转染GLUT3融合蛋白的PC12细胞相似。这些研究首次检测了PC12细胞中GLUT3的运输和融合，并建立了一个研究神经元葡萄糖转运蛋白调节的模型系统。
